Lack of correlation between placental gene expression and RNA integrity number (RIN) or time to collection

Placenta ◽  
2014 ◽  
Vol 35 (9) ◽  
pp. A46-A47 ◽  
Author(s):  
Christopher J. Stodgell ◽  
Richard K. Miller ◽  
Linda Salamone ◽  
Jeffrey Murray ◽  
Jia Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Integrity ◽  
Rna Integrity Number

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

2018 ◽  
Author(s):  
M Grinstein ◽  
HL Dingwall ◽  
RR Shah ◽  
TD Capellini ◽  
JL Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Biological Variation ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number

AbstractBackgroundMechanistic understanding of tendon molecular and cellular biology is crucial towards furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between 3-5 months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RNA-seq and RT-qPCR. We show this can reduce biological variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues as well as precious human samples.

150 Effect of bovine seminal plasma and sperm on endometrial gene expression

2019 ◽  
Vol 31 (1) ◽  
pp. 200
Author(s):  
B. Fernandez-Fuertes ◽  
J. M. Sanchez ◽  
S. Bages ◽  
P. Lonergan
Keyword(s):  
Gene Expression ◽  
Seminal Plasma ◽  
Reproductive Tract ◽  
Epididymal Sperm ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Maternal Immune System ◽  
Negative Effect

In cattle, most pregnancy losses are sustained before implantation. Many factors are involved in implantation failure, but in mice, pigs and humans there is increased evidence of a role for the maternal immune system and its regulation by seminal plasma (SP). However, there is little evidence for a role of SP in bovine fertility, where dilution or removal of SP before AI is routine. Therefore, the aim of this work was to determine the effect of bull SP or sperm on endometrial gene expression. To this end, 6 heifers were oestrous synchronised and slaughtered 12h after the onset of oestrus. Five endometrial explants from the horn ipsilateral to the preovulatory follicle were obtained from each animal. Explants were incubated with (1) RPMI medium (control); (2) epididymal sperm (106 epididymal sperm mL−1); (3) complete ejaculate (106 ejaculated sperm mL−1+25% SP); (4) ejaculated sperm alone (106 ejaculated sperm mL−1); and (5) SP alone (25% SP). Epididymal sperm were collected and pooled from the cauda epididymis of 3 bulls slaughtered in a commercial abattoir. In addition, complete ejaculates were obtained from 3 other bulls using an artificial vagina. After pooling the samples, a small volume was washed through a density gradient to obtain the ejaculated sperm, and the rest of the ejaculate was filtered to obtain sperm-free SP. The RPMI media was used to dilute sperm and SP to the working concentrations. After 6h of incubation, explants were snap frozen. The RNA quality was assessed with an Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA) before RT-qPCR analysis. Interestingly, SP had a dramatic effect on endometrium RNA integrity, as evidenced by a lower RNA integrity number in explants exposed to a complete ejaculate (2.4±0.14) or SP (2.4±0.06), in comparison with the control, epididymal sperm, or ejaculated sperm treatments (6.7±0.43, 6.9±0.32, 6.7±0.30, respectively; P<0.05). Due to the low RNA quality, those treatments were excluded from further analysis. However, this finding is currently being explored, along with the possibility of this effect being inherent to species that ejaculate intravaginally. We then compared the ability of ejaculated sperm (which have been exposed to SP) and epididymal sperm (which have never had contact with SP) to regulate the endometrial expression of IL1A, IL1B, IL8, IL6, PTGES2, TNFA, and LIF. Although IL6, IL1A and LIF increased in all animals when exposed to either ejaculated or epididymal sperm, there was no effect of treatment. In conclusion, these data did not support the notion that exposure of sperm to SP is important for the immune regulation of the bovine uterus. In addition, the negative effect of SP on the endometrium, together with the fact that bulls are intravaginal ejaculators, suggests that any putative immunoregulatory role of this fluid in the cattle uterus is indirect. Further analysis of the effect of SP on the vagina and cervix will help elucidate whether this response is present in this species and whether it can propagate to more distal regions of the reproductive tract. This work was supported by Science Foundation Ireland (13/IA/1983, 16/IA/4474).

scholarly journals The spatial RNA integrity number assay for in situ evaluation of transcriptome quality

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Linda Kvastad ◽  
Konstantin Carlberg ◽  
Ludvig Larsson ◽  
Eva Gracia Villacampa ◽  
Alexander Stuckey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Spatial Variation ◽  
Tissue Quality ◽  
Rna Integrity ◽  
Quality Metric ◽  
Rna Quality ◽  
Rna Integrity Number ◽  
Cellular Resolution

AbstractThe RNA integrity number (RIN) is a frequently used quality metric to assess the completeness of rRNA, as a proxy for the corresponding mRNA in a tissue. Current methods operate at bulk resolution and provide a single average estimate for the whole sample. Spatial transcriptomics technologies have emerged and shown their value by placing gene expression into a tissue context, resulting in transcriptional information from all tissue regions. Thus, the ability to estimate RNA quality in situ has become of utmost importance to overcome the limitation with a bulk rRNA measurement. Here we show a new tool, the spatial RNA integrity number (sRIN) assay, to assess the rRNA completeness in a tissue wide manner at cellular resolution. We demonstrate the use of sRIN to identify spatial variation in tissue quality prior to more comprehensive spatial transcriptomics workflows.

scholarly journals A robust method for RNA extraction and purification from a single adult mouse tendon

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4664 ◽  
Author(s):  
Mor Grinstein ◽  
Heather L. Dingwall ◽  
Rishita R. Shah ◽  
Terence D. Capellini ◽  
Jenna L. Galloway
Keyword(s):  
Gene Expression ◽  
Adult Mouse ◽  
Rna Extraction ◽  
Cost Effective ◽  
Mouse Genetics ◽  
Rna Integrity ◽  
Rna Quality ◽  
Rna Analysis ◽  
Rna Integrity Number ◽  
Extraction And Purification

BackgroundMechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts.MethodsSingle Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed.ResultsAfter testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis ofScxgene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyzeSox9andCol1a2gene expression changes in injured compared with uninjured control tendons.DiscussionOur work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

Preanalytical robustness of blood collection tubes with RNA stabilizers

2019 ◽  
Vol 57 (10) ◽  
pp. 1522-1529 ◽  
Author(s):  
Chiara Stellino ◽  
Gaël Hamot ◽  
Camille Bellora ◽  
Johanna Trouet ◽  
Fay Betsou
Keyword(s):  
Storage Temperature ◽  
Regulation Of Gene Expression ◽  
Analysis Data ◽  
Blood Collection ◽  
Becton Dickinson ◽  
Rna Integrity ◽  
Qrt Pcr ◽  
Rna Integrity Number ◽  
Significant Difference ◽  
Blood Collection Tubes

Abstract Background Efficient blood stabilization is essential to obtaining reliable and comparable RNA analysis data in preclinical operations. PAXgene (Qiagen, Becton Dickinson) and Tempus (Applied Biosystems, Life Technologies) blood collection tubes with RNA stabilizers both avoid preanalytical degradation of mRNA by endogenous nucleases and modifications in specific mRNA concentrations by unintentional up- or down-regulation of gene expression. Methods Sixteen different preanalytical conditions were tested in PAXgene and Tempus blood samples from seven donors: different mixing after collection, different fill volumes and different 24-h transport temperature conditions after collection. RNA was extracted by column-based methods. The quality of the extracted RNA was assessed by spectrophotometric quantification, A260/A280 purity ratio, RNA Integrity Number (Agilent Bioanalyzer), miRNA quantative real time polymerase chain reaction (qRT-PCR) on two target miRNAs (RNU-24 and miR-16), mRNA quality index by qRT-PCR on the 3′ and 5′ region of the GAPDH gene, and the PBMC preanalytical score, based on the relative expression levels of the IL8 and EDEM3 coding genes. Results When PAXgene RNA and Tempus blood collection tubes were used following the manufacturers’ instructions, there was no statistically or technically significant difference in the output RNA quality attributes. However, the integrity of the RNA extracted from Tempus collection tubes was more sensitive to fill volumes and effective inversion, than to storage temperature, while the integrity of RNA extracted from PAXgene collection tubes was more sensitive to effective inversion and storage temperature than to fill volumes. Conclusions Blood collection tubes with different RNA stabilizers present different robustness to common preanalytical variations.

Stress–response relationships related to ageing and death of orthodox seeds: a study comparing viability and RNA integrity in soya bean (Glycine max) cv. Williams 82

2020 ◽  
Vol 30 (2) ◽  
pp. 161-172
Author(s):  
Christina Walters ◽  
Margaret B. Fleming ◽  
Lisa M. Hill ◽  
Emma J. Dorr ◽  
Christopher M. Richards
Keyword(s):  
Degradation Kinetics ◽  
Rna Degradation ◽  
Soya Bean ◽  
Rna Integrity ◽  
Viability Loss ◽  
Rna Integrity Number ◽  
Oxidative Stresses ◽  
Degradation Rates ◽  
Highly Correlated ◽  
Kinetics Of

AbstractCharacterizing non-lethal damage within dry seeds may allow us to detect early signs of ageing and accurately predict longevity. We compared RNA degradation and viability loss in seeds exposed to stressful conditions to quantify relationships between degradation rates and stress intensity or duration. We subjected recently harvested (‘fresh’) ‘Williams 82’ soya bean seeds to moisture, temperature and oxidative stresses, and measured time to 50% viability (P50) and rate of RNA degradation, the former using standard germination assays and the latter using RNA Integrity Number (RIN). RIN values from fresh seeds were also compared with those from accessions of the same cultivar harvested in the 1980s and 1990s and stored in the refrigerator (5°C), freezer (−18°C) or in vapour above liquid nitrogen (−176°C). Rates of viability loss (P50−1) and RNA degradation (RIN⋅d−1) were highly correlated in soya bean seeds that were exposed to a broad range of temperatures [holding relative humidity (RH) constant at about 30%]. However, the correlation weakened when fresh seeds were maintained at high RH (holding temperature constant at 35°C) or exposed to oxidizing agents. Both P50−1 and RIN⋅d−1 parameters exhibited breaks in Arrhenius behaviour near 50°C, suggesting that constrained molecular mobility regulates degradation kinetics of dry systems. We conclude that the kinetics of ageing reactions at RH near 30% can be simulated by temperatures up to 50°C and that RNA degradation can indicate ageing prior to and independent of seed death.

scholarly journals The kinetics of ageing in dry-stored seeds: a comparison of viability loss and RNA degradation in unique legacy seed collections

2018 ◽  
Vol 123 (7) ◽  
pp. 1133-1146 ◽  
Author(s):  
Margaret B Fleming ◽  
Lisa M Hill ◽  
Christina Walters
Keyword(s):  
Storage Time ◽  
Storage Temperature ◽  
Rna Degradation ◽  
Seed Longevity ◽  
Rna Integrity ◽  
Viability Loss ◽  
Rna Integrity Number ◽  
Seed Ageing ◽  
Mode Of Detection ◽  
Ageing Rate

Abstract Background and Aims Determining seed longevity by identifying chemical changes that precede, and may be linked to, seed mortality, is an important but difficult task. The standard assessment, germination proportion, reveals seed longevity by showing that germination proportion declines, but cannot be used to predict when germination will be significantly compromised. Assessment of molecular integrity, such as RNA integrity, may be more informative about changes in seed health that precede viability loss, and has been shown to be useful in soybean. Methods A collection of seeds stored at 5 °C and 35–50 % relative humidity for 1–30 years was used to test how germination proportion and RNA integrity are affected by storage time. Similarly, a collection of seeds stored at temperatures from −12 to +32 °C for 59 years was used to manipulate ageing rate. RNA integrity was calculated using total RNA extracted from one to five seeds per sample, analysed on an Agilent Bioanalyzer. Results Decreased RNA integrity was usually observed before viability loss. Correlation of RNA integrity with storage time or storage temperature was negative and significant for most species tested. Exceptions were watermelon, for which germination proportion and storage time were poorly correlated, and tomato, which showed electropherogram anomalies that affected RNA integrity number calculation. Temperature dependencies of ageing reactions were not significantly different across species or mode of detection. The overall correlation between germination proportion and RNA integrity, across all experiments, was positive and significant. Conclusions Changes in RNA integrity when ageing is asymptomatic can be used to predict onset of viability decline. RNA integrity appears to be a metric of seed ageing that is broadly applicable across species. Time and molecular mobility of the substrate affect both the progress of seed ageing and loss of RNA integrity.

Studies on RNA integrity and gene expression in human myocardial tissue, pericardial fluid and blood, and its postmortem stability

2013 ◽  
Vol 232 (1-3) ◽  
pp. 218-228 ◽  
Author(s):  
Lucas González-Herrera ◽  
Aurora Valenzuela ◽  
Juan A. Marchal ◽  
José A. Lorente ◽  
Enrique Villanueva
Keyword(s):  
Gene Expression ◽  
Pericardial Fluid ◽  
Myocardial Tissue ◽  
Rna Integrity ◽  
Postmortem Stability

The Influence of Tissue Procurement Procedures on RNA Integrity, Gene Expression, and Morphology in Porcine and Human Liver Tissue

2015 ◽  
Vol 13 (3) ◽  
pp. 200-206 ◽  
Author(s):  
Marcel Kap ◽  
Anieta M. Sieuwerts ◽  
Mikael Kubista ◽  
Monique Oomen ◽  
Shazia Arshad ◽  
...  
Keyword(s):  
Gene Expression ◽  
Liver Tissue ◽  
Human Liver ◽  
Human Liver Tissue ◽  
Rna Integrity ◽  
Tissue Procurement
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